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Team:Cornell/Week 7
From 2011.igem.org
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==Wednesday, July 20== | ==Wednesday, July 20== | ||
Lab work done by: James Mathew | Lab work done by: James Mathew | ||
| - | *'''Set up ligation reaction of pZE-12 backbone with the primer dimer insert | + | *'''Set up ligation reaction of pZE-12 backbone with the primer dimer insert''' |
::1. <u>Digestion of Backbone</u> | ::1. <u>Digestion of Backbone</u> | ||
| - | :::- | + | :::- 26µl Backbone plasmid = 2µg DNA |
| - | :::- | + | :::- 5µl NEBuffer 4 (optimal for SphI-HF and ClaI-HF) |
| - | :::- | + | :::- 1µl SphI & 1µl ClaI |
| - | :::- 16. | + | :::- 16.5µl H2O |
::::*left in water bath for one hour | ::::*left in water bath for one hour | ||
| - | :::- | + | :::- 1µl CIAP added to digestion reaction |
::::*left in water bath for 30 minutes | ::::*left in water bath for 30 minutes | ||
| - | |||
::2. <u>Ran Digestion Product through gel for 30 minutes at 120 V.</u> | ::2. <u>Ran Digestion Product through gel for 30 minutes at 120 V.</u> | ||
| - | ::::Lane 1: | + | ::::Lane 1: 2µl 1kb Ladder + 2µl 6X loading dye + 8µl H2O |
| - | ::::Lane 2: | + | ::::Lane 2: 25µl digestion reaction + 5µl 6X loading dye |
| - | ::::Lane 3: | + | ::::Lane 3: 25µl digestion reaction + 5µl 6X loading dye |
| - | + | ||
::3. <u>Gel Purification using Qiagen Kit</u> | ::3. <u>Gel Purification using Qiagen Kit</u> | ||
| - | |||
::4. <u>NanoDrop of Samples</u> | ::4. <u>NanoDrop of Samples</u> | ||
| - | |||
| - | |||
| - | |||
::5. <u>Ligation Reaction</u> | ::5. <u>Ligation Reaction</u> | ||
| - | : | + | :*Ligation done overnight for transformation on 7/21/11 |
| - | + | ||
| - | *Ligation done overnight for transformation on 7/21/11 | + | |
==Thursday, July 21== | ==Thursday, July 21== | ||
Revision as of 01:58, 16 September 2011
Week 1 | Week 2 - 5 | Week 6 | Week 7 | Week 8 | Week 9 | Week 10 | Week 11 | Week 12 | Week 13 | Week 14 | Week 15 | Week 16 | Week 17 | Week 18 | Week 19 | Week 20 | Week 21 |
July 17th - July 23rd
Sunday, July 17
Lab work done by: Alyssa Henning, Bill Jo
- Miniprepped 1 sample of GFP + AviTag (the second sample got messed up)
- Ran a gel on the RFP that was amplified via PCR on Saturday
- Gel purification and extraction
- Sean will inoculate 6 tubes of GFP-containing bacteria -- 1 tube for us, 5 tubes for CURIE (Cornell iGEM's outreach program)
Monday, July 18
Lab work done by: Charlie Chung, Youjin Cho
- Gel purified 2 RFP PCR samples from Friday
- Miniprep 2 samples of GFP + AviTag
Tuesday, July 19
Lab work done by: James Mathew
- Submitted GFP + AviTag for synthesis
- Submitted pZE12 backbone for subcloning of light sensor
Wednesday, July 20
Lab work done by: James Mathew
- Set up ligation reaction of pZE-12 backbone with the primer dimer insert
- 1. Digestion of Backbone
- - 26µl Backbone plasmid = 2µg DNA
- - 5µl NEBuffer 4 (optimal for SphI-HF and ClaI-HF)
- - 1µl SphI & 1µl ClaI
- - 16.5µl H2O
- left in water bath for one hour
- - 1µl CIAP added to digestion reaction
- left in water bath for 30 minutes
- 2. Ran Digestion Product through gel for 30 minutes at 120 V.
- Lane 1: 2µl 1kb Ladder + 2µl 6X loading dye + 8µl H2O
- Lane 2: 25µl digestion reaction + 5µl 6X loading dye
- Lane 3: 25µl digestion reaction + 5µl 6X loading dye
- 3. Gel Purification using Qiagen Kit
- 4. NanoDrop of Samples
- 5. Ligation Reaction
- Ligation done overnight for transformation on 7/21/11
- 1. Digestion of Backbone
Thursday, July 21
Lab work done by: James Mathew
- miniprepped Vio operon (Cambridge 2009)
Lab work done by: Youjin Cho, Charlie Chung, and Alyssa Henning
- Reconstituted VioA, VioB, and VioE forward and reverse primers
- Set up PCR for VioA, VioB, and VioE
- Aborted transformation of pZE-12 plasmid + avitag primer dimer because reverse primers for avitag were incorrect.
- Redesigned reverse primers for avitag to order on Friday
Friday, July 22
Lab work done by: James Mathew & Claire Paduano
- PCR gel purification of VioA, VioB, and VioE
Saturday, July 23
Lab work done by: James Mathew & Claire Paduano
- Digestion of VioA, VioB, VioE, GFP, and RFP in preparation for ligation with corrected backbone and avitag primer dimer
